The hepatitis B virus (HBV) belongs to the Hepadnaviridae, a group of hepatotropic DNA viruses. This group of viruses comprises not only human or primate HBV, but also duck-, woodchuck-, ground squirrel-, tree squirrel- and heron HBV. The genome of human HBV consists of a partially double-stranded ˜3.2 kb DNA molecule. The HBV genome contains four partly overlapping open reading frames (ORFs) being (i) the preC/C ORF encoding the secreted e antigen (HBeAg) and nucleocapsid core protein (HBcAg), respectively; (ii) the P ORF encoding the viral polymerase/reverse transcriptase; (iii) the preS1/preS2/S ORF encoding the viral envelope proteins, large, middle and small s antigen (HBsAg), respectively; and (iv) the X ORF encoding a transcriptional trans-activator protein.
The HBV envelope comprises three related glycoproteins, termed hepatitis B surface antigens (HBsAg), which are the product of the S gene: 1) the “small” transmembrane protein, Also termed major protein or small S-protein, composed of 226 amino acids (aa), 2) the “middle” protein which comprises the small S-protein and 55 additional aa at the N-terminus corresponding to the pre-S2 region of the S gene, and 3) the “large” protein composed of 389 (genotype D), 399 (genotypes E and G) or 400 (genotypes A to C and F) aa corresponding to the following regions: S+pre-S2+pre-S1 (108-119 N-terminal aa) (Robinson et al., 1987; Heermann et al., 1984). The envelope of HDV (hepatitis delta virus) is also entirely derived from HBV and consists predominantly of small HBsAg, 5-10% of middle HBsAg and no or less then 1% of large HBsAg (Bonino et al., 1986).
Hepatitis B viruses exhibit a large genetic variability in their genomes, with currently 7 HBV genotypes (A to G) being recognized (Stuyver et al., 2001; Stuyver et al., 2000). Furthermore, hepatitis B viruses do not circulate in infected individuals as homogeneous populations of identical viral particles, but rather as a pool of genetically distinct but closely related variants. This genetic variation confers a significant advantage to the virus, as the simultaneous presence of multiple variant genomes and the high rate at which new variants are generated allow rapid selection of mutants better suited to survive in new environmental conditions. Mutations in the HBV polymerase and their clinical relevance are discussed infra.
Intervention strategies to control the burden of chronic liver disease caused by HBV include primary prevention through vaccination and chemoprevention through antiviral therapy (Hoofnagle et al., 1993). Both of these strategies provide powerful selection pressures, which can result in the emergence of variant viruses or “escape” mutants. Such mutants may emerge as a function of four factors: the viral mutation frequency, the intrinsic mutability of the antiviral target site, the selective pressure and the magnitude and rate of virus replication (Richman, 1996).
Until recently, the only licensed treatment for chronic hepatitis B was interferon-alpha (IFN-α), which proves to be partially effective only in a small group of carriers (Lok, 1994). This relative failure of IFN-α for the treatment of chronic HBV infection has prompted the search for further therapeutic agents and regimes. In particular, a number of nucleoside analogues have been shown to inhibit hepadnaviral replication via inhibition of the hepadnaviral DNA polymerase/reverse transcriptase. Some of these compounds have already been withdrawn from clinical use due to toxicity (lobucavir) or lack of efficacy (famciclovir) (De Clercq, 1999; Schinazi, 1997; Luscombe et al., 1996). On the other hand, other compounds such as Adefovir Dipivoxil (Gilead Sciences) and Entecavir (Bristol Myers Squibb) show much promise and are currently in phase III clinical trials. At this moment, the most successful nucleoside analogue is without doubt lamivudine (Jarvis et al., 1999), which was recently licensed. Lamivudine is a deoxycytidine analogue, which is phosphorylated by histidine kinases to a triphosphate moiety that is active against HBV (Wang et al., 1998). Lamivudine inhibits HBV replication, reduces hepatic necroinflammatory activity and the progression of fibrosis in patients with chronic hepatitis B (Yao et al., 1999; Lai et al., 1998; Dienstag et al., 1998; Schiff et al., 1998). Lamivudine also reduces ongoing viral replication and compensated liver disease including HBe-Ag negative patients (Tassopoulos et al., 1999). The drug also suppresses viral replication in liver transplant recipients and HIV-positive patients (Markowitz et al., 1998; Wright et al., 1997; Benhamou et al., 1996). However, lamivudine-resistant DNA polymerase variants have been isolated from patients with chronic hepatitis B during treatment with lamivudine (Hunt et al., 2000; Nafa et al., 2000; Yeh at al., 2000; Ling et al., 1999) and references therein). One year of treatment with lamivudine (100 mg daily) results in the appearance of the ‘YVDD’ mutation (wild-type motif is ‘YMDD’) in the DNA polymerase in 14-43% of HBV-infected patients (Dienstag et al., 1999; Lai et al., 1998). The mutation rate increases with prolonged use of lamivudine (Liaw et al., 2000). Another lamivudine-induced mutation turns the YMDD motif into YIDD. Both mutations of the amino acid residue 552 (see, however, Table 1 for the HBV genotype-dependent numbering of the amino acid residues) in the C-domain of the HBV DNA polymerase/reverse transcriptase (i.e. M552V and M552I) can occur in combination with another mutation in the B-domain of the HBV DNA polymerase/reverse transcriptase, namely the L528M mutation. The M552V and M552I mutations alone, as well as combination of either one with the L528M mutation have been shown to confer resistance of HBV replication to lamivudine and famciclovir (Ono et al., 2001; Xiong et al., 2000; Delaney et al., 2000; Ono-Nita et al., 2000; Fu et al., 1999; Xiong et al., 1998). Another associated mutation is the V/L/M555I mutation which, either alone or in combination with M552I is conferring low resistance of HBV replication to lamivudine or famciclovir (Fu et al., 1999).
Both in vitro and in vivo studies have demonstrated that YMDD variants, i.e. HBV variants comprising ‘YVDD’ or ‘YIDD’ in the C-domain of the HBV DNA polymerase/reverse transcriptase are less replication competent compared to the wild-type, are associated with lower HBV DNA levels compared to pretreatment levels, and can be associated with continued histologic improvement (Leung, 2000; Ling et al., 1999; Ono-Nita et al., 1999). However, said YMDD variants have also been reported to cause hepatic decompensation (Liaw et al., 1999). As for the limited studies completed at this moment, lamivudine-resistant HBV does not confer cross-resistance to adefovir (Xiong et al., 1998).
At least some of the lamivudine-induced mutations appearing in the HBV DNA polymerase also occur after prolonged treatment with famciclovir and are described in e.g. Bartholomeusz et al. (1998) (International Patent Publication Number WO 98/21317) and Bartholomeusz et al. (2000) (International Patent Publication Number WO 00/61758). A comprehensive review of HBV resistance to antiviral drugs is given in (Delaney et al., 2001).
Key mutations involved in lamivudine-resistance of the HBV DNA polymerase/transcriptase have been identified as L180M, M204V/I and MN/L207I (see, however, Table 1 for HBV genotype-dependent numbering of amino acid 204 in the HBV DNA polymerase/reverse transcriptase).
It is known in the art that HIV (human immunodeficiency virus) also contains the YMDD motif in its reverse transcriptase domain. By means of in vitro mutagenesis, said motif of the HIV reverse transcriptase has been converted into YSDD. The resulting mutant HIV reverse transcriptase was only 5 to 10% as active in vitro as the wild-type HIV reverse transcriptase (Wakefield et al., 1992). In the same study, however, it is not at all mentioned that said mutation could be induced by treatment of a HIV-infected patient with an antiviral drug, i.e. could occur in vivo. Nor is it mentioned that the YMDD motif is part of the HBV DNA polymerase/reverse transcriptase. The occurrence of the YSDD mutation in the HBV DNA polymerase during lamivudine-treatment of a HBV-infected patient was described by Bozdayi et al. (Bozdayi et al., 2001).